1. Field of the Invention
This invention relates to thromboplastin extracts and reagents, and more particularly to thromboplastin extracts and reagents including a chelator.
2. Technology Background
Blood coagulation involves a succession of reactions leading to the formation of fibrin. Fibrin is the protein that holds blood clots together. Fibrin is formed from a soluble precursor, fibrinogen, by the protease thrombin. Thrombin is generated during clotting from an inactive precursor, prothrombin (Factor II), through one of two mechanisms known as the extrinsic and intrinsic pathways. The extrinsic pathway comes into action in response to tissue injury, and involves the participation of blood clotting factors V, VII, and X. The extrinsic and intrinsic pathways converge in a final common pathway for the thrombin-catalyzed conversion of fibrinogen to the fibrin clot.
Clinical tests of blood coagulation include the prothrombin time test. The prothrombin time test is a measure of the extrinsic pathway of blood coagulation. Prothrombin time, hereinafter (PT), measures the time elapsing until plasma clots in the presence of a tissue thromboplastin reagent. The test is performed by adding a tissue thromboplastin reagent to the plasma and determining the time necessary for coagulation. The thromboplastin reagent, along with factor VII, activates factor X to X.sub.a (active factor X) in the presence of factor V.sub.a (active factor V), Ca.sup.2+, and platelet phospholipids. Prothrombin is converted to thrombin, which in turn catalyzes the formation of fibrin from fibrinogen. Thus, a deficiency of factor II, V, VII, or X or a severe deficiency of fibrinogen will prolong the PT. Normal plasma contains factors VII and X; therefore, if normal plasma corrects the PT, the defect must be due to a deficiency of one of those two factors.
Blood coagulation requires calcium. Since calcium is present in plasma itself, anticoagulants such as sodium citrate or EDTA are often placed in the vials that hold plasma to block its spontaneous coagulation. The plasma together with sodium citrate or EDTA as an anticoagulant is referred to as anticoagulated plasma. Calcium ions in a saturating amount are added according to the prior art to overcome the sodium citrate or EDTA and to prevent the sodium citrate or EDTA from affecting PT time.
The prothrombin time test can monitor coumarin therapy for maintenance of the therapeutic range. The coumarin drugs are used clinically to impede blood coagulation in thrombotic states, including heart disease. They inhibit the vitamin K-dependent synthesis of factors II, VII, IX and X.
U.S. Pat. No. 3,522,148, dated Jul. 28, 1970, to Adam, Jr. discloses a stabilized thromboplastin reagent including a mixture of a saline extract of acetone-dried rabbit brain tissue and a sodium salt, and an equal volume of aqueous solution of calcium chloride. Adam, Jr. also discloses a stabilized thromboplastin reagent including a mixture of a saline extract of acetone-dried rabbit brain tissue and an equal volume of a calcium salt of a sugar acid. U.S. Pat. No. 4,784,944, dated Nov. 15, 1988, to Kolde, H. J. discloses thromboplastin obtained from human placenta.
The three general steps in preparing a thromboplastin reagent include preparing a powdered thromboplastin source, converting the powdered thromboplastin source into a thromboplastin extract, and preparing a useful thromboplastin reagent from the thromboplastin extract.
Sources of thromboplastin include rabbit brain, human placenta, bovine brain, ox brain, human brain, and thromboplastin produced by recombinant DNA technology. Powdered thromboplastin sources include rabbit brain powder, powdered human placenta, powdered bovine brain, powdered ox brain, powdered human brain, and powdered thromboplastin produced by recombinant DNA technology.
Rabbit brain powder is prepared according to the prior art by homogenizing whole rabbit brains stripped of attached blood vessels with excess acetone, and drying the slurry under vacuum. Rabbit brain powder is commercially available.
Powdered human placenta is prepared according to the prior art in a manner similar to the preparation of rabbit brain powder.
Rabbit brain powder is converted into a useful thromboplastin extract according to the prior art by a procedure that involves extraction of the powder in warm saline solution and centrifugation to remove the sedimented brain powder, leaving a supernatant extract rich in thromboplastin. U.S. Pat. No. 4,416,812, dated Nov. 22, 1983, to Becker et al. discloses using calcium ions during extraction.
The thromboplastin reagent is then prepared from the extract by combining the extract with stabilizers and preservatives and one of various calcium salts to produce a bulk reagent, and then usually lyophilizing the reagent for long-term storage.
A problem which limits the effectiveness of the prothrombin time test is that chemical variation of the thromboplastin extract and the thromboplastin reagent formulations can alter the PT. The PT for normal human plasma based on the use of fresh basic raw material, rabbit brain powder, is the standard value for comparison. Physicians in the United States relying on PT results for screening and diagnostic purposes generally expect normal human plasma to clot within 12 seconds after contact with a standardized thromboplastin reagent that is lyophilized and reconstituted. The in-process extract solutions before lyophilization and reconstitution into a standardized thromboplastin reagent are required to provide a PT of less than or equal to 11 seconds. From time to time, for as yet unknown reasons, rabbit brain powder from commercial suppliers may vary from its normal quality, and produce extracts unsuitable for reagent preparation by conventional methods because they yield long PTs for normal human plasma.
One way to accomplish standardized results of the prothrombin time test is to provide extracts and reagents that have a consistently lower PT of normal human plasma from lot to lot. Another way to achieve comparability is standardization in reference to a particular factor sensitivity. "Sensitive" reagents need not provide a lower PT value. However, it is beneficial for even so-called "sensitive" reagents to provide a lower PT value so that the effects of the variables that are modified to achieve a particular factor sensitivity do not prolong the reagent's PT of normal human to so great an extent that testing becomes impractical with automated instrumentation.
Accordingly, it is the main object of the present invention to provide a process for preparing a thromboplastin extract and reagent which overcomes the aforementioned disadvantages. Specifically, it is a principal object of this invention to provide extract and a thromboplastin reagent that consistently provides a lower PT of normal human plasma from lot to lot.
Another object of the invention is to improve a wide variety of powdered thromboplastin sources within a species such as rabbit brain powders all to a standard level of PT, thus allowing for improved lot-to-lot thromboplastin reagent reproducibility, so that data can be compared for meaningful, consistent results.
Another object of the invention is to provide a thromboplastin reagent from a raw powdered thromboplastin source such as rabbit brain powder that may not otherwise be useful for preparing a standardized thromboplastin reagent.
These objects and others which will become apparent as the specification progresses are accomplished by the invention.